CN1772920A - Fast and sensitive method of detecting virus infection titer of attenuated live hepatitis A vaccine - Google Patents

Fast and sensitive method of detecting virus infection titer of attenuated live hepatitis A vaccine Download PDF

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CN1772920A
CN1772920A CNA2005100110869A CN200510011086A CN1772920A CN 1772920 A CN1772920 A CN 1772920A CN A2005100110869 A CNA2005100110869 A CN A2005100110869A CN 200510011086 A CN200510011086 A CN 200510011086A CN 1772920 A CN1772920 A CN 1772920A
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virus
hepatitis
primer
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hav
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CN100389207C (en
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孙明波
蒋燕军
廖国阳
李卫东
周健
姜述德
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Institute of Medical Biology of CAMS and PUMC
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Abstract

The present invention provides one kind of fast and sensitive method for detecting virus infection titer of attenuated live hepatitis A vaccine. The method determines the existence of the virus via detecting the negative brand RNA intermediate of marker appearing during duplicating hepatitis A virus, and is superior to available inverse transcription PCR method and cell culturing method. The method has culture period shortened to 8 days, short detection period, no need of replacing maintaining liquid, less contamination possibility, less cell post-treating steps, high work efficiency and capacity of providing virus heredity information via sequencing the PCR product.

Description

The method of Sensitive Detection virus infection titer of attenuated live hepatitis A vaccine fast,
Technical field
The present invention relates to a kind of method that hepatitis A virus (HAV) is detected, belong to medical biotechnology field.
Background technology
Hepatitis A (to call hepatitis A in the following text) is a kind of world wide popular common disease toxicity infectious intestinal disease, has 1,500,000 clinical case appearance every year at least.At present, the popular situation of China's hepatitis A is still very severe, and fresh condition occurred.In most of area, sanitary condition still falls behind, and belongs to the hepatitis A hotspot on the one hand, during the outburst of group hepatitis A report is arranged, and national hepatitis A sickness rate is about 16,/10 ten thousand.Show that according to the Ministry of Health's announcement data first quarter in 2004, hepatitis A occupies one of preceding 5 transmissible diseases.In the developed area,, at the age of population infection hepatitis A, pass in early days to more than the adolescence on the other hand by children along with the improvement of sanitary condition.Infect without hepatitis A, the Susceptible population that does not form hepatitis A antibody in the body constantly accumulates.In addition, developed regions population from other places's increase and population mobility's increase have more increased the spread scope and the speed of hepatitis A.In a word, China just transforms from the high popular area of hepatitis A to the popular area of moderate at present, but high, in the staggered form that exists in popular district with long-term existence, be characterized in that hepatitis A infected to the big age to pass, Susceptible population constantly accumulates, the dangerous thereby increase of hepatitis A popular.Using hepatitis A vaccine to carry out active immunity is the main means of control hepatitis A popular.Present stage, China mainly uses Attenuated HAV to carry out immunoprophylaxis.
The method that detects hepatitis A virus (HAV) at present mainly contains two kinds: one, cell culture method, this method are earlier viral sample after for some time, to be detected hepatitis A virus (HAV) antigen with the immunology means with the sensitive cells separation and Culture again, whether contain virus in the judgement sample.But the advantage of cell culture method is an infectious virus in the indirect detection sample, and can carry out quantitatively virus infection titer.But hepatitis A virus (HAV) does not generally cause the host cell pathology, poor growth, and the cycle is long, and the malicious incubation time of best product needed for 3 to 4 weeks, and viral yield is not high.These growth characteristics can not with as detect the plaque method of infectious poliovirus or cytopathy and test and detect hepatitis A virus (HAV), carry out immunology detection and must depend on the specific antibody behind the mark, make that simultaneously cell culture method testing amount is big, cycle is tediously long, often needs more than one month.Two, molecular biology method, common molecular biology method are the reverse transcriptase polymerase chain reaction methods, and direct hepatitis A virus (HAV) positive chain RNA in the test sample is for the situation that exists of hepatitis A virus (HAV) provides reliable and direct evidence.The reverse transcriptase polymerase chain reaction method is highly sensitive, and specificity is good, and the operating time is short, and can be by order-checking provides viral genetic information to the polymerase chain reaction product.But reverse transcriptase polymerase chain reaction has a major defect, promptly can not distinguish infectious virus and inactivation virus.As can be seen, this two classes detection method is to judge the existence of virus by detecting hepatitis A virus (HAV) self inherent structure such as antigen protein or genome positive chain RNA.
It is an important indicator of Attenuated HAV quality control that virus infection titer detects.At present, the main virus infection titer that adopts cell culture method (CCID50) to carry out Attenuated HAV detects (" Chinese biological goods rules ", 2000 editions), its process is done 10 times of dilutions for getting vaccine sample, is taken to few 3 extent of dilution and inoculates human diploid cell respectively, be cultured to the virus multiplication peak at 35 ℃, harvested cell, through multigelation, virus antigen is extracted in ultrasonic back, detect the hepatitis A virus (HAV) antigen protein with the ELISA method, judge virus infection titer.This method steps is numerous and diverse, and is consuming time longer, and the time needed more than 4 weeks, often causes vaccine staging life prolongation in the storehouse, and reality has shortened the validity period of vaccine.
The hepatitis A virus gene group is made of single positive chain RNA.When duplicating, hepatitis A virus gene group positive chain RNA is at first transcribed out strand RNA, is the synthetic newborn positive chain RNA of future generation of template again with the minus strand.Strand RNA occurs in back two days of infection, peaks to the 8th day, and the 10th day to 14 days begin to fall after rise.Can find out that thus the appearance of strand RNA is the sign that hepatitis A virus (HAV) is duplicated, also be the direct evidence that infectious hepatitis A virus (HAV) exists.
Summary of the invention
Numerous and diverse for overcoming the step that has the cell culture method existence now, consuming time longer, often cause the vaccine stock phase long, shorten deficiencies such as vaccine validity period, and the reverse transcriptase polymerase chain reaction method exist can not distinguish deficiencies such as infectious virus and inactivation virus, the invention provides a kind of method of quick, special and Sensitive Detection virus infection titer of attenuated live hepatitis A vaccine.
The molecular mechanism that the present invention duplicates according to hepatitis A virus (HAV), adopt the label type reverse transcriptase polymerase chain reaction, detect the mark strand RNA intermediate that occurs when hepatitis A virus (HAV) is duplicated, and judge existing of infectious virus with this, by software analysis and experimental verification, determine primer and each reaction parameter of reverse transcriptase polymerase chain reaction, thereby rapid detection goes out the existence of infectious hepatitis A virus (HAV), and calculate the vaccine virus infection titre.
The present invention realizes by following concrete technical scheme: a kind of fast, the method for Sensitive Detection virus infection titer of attenuated live hepatitis A vaccine, it is characterized in that comprising the following steps:
A, virus culture
The KMB17 cell is implanted in the little square vase, be cultured to grow up to fresh individual layer after, the Attenuated HAV sample is made 10 times of serial dilutions, getting extent of dilution is 10 -4-10 -9Viral liquid be seeded on this cell monolayer, adsorb after 1～2 hour, add and keep liquid, cultivated 5-8 days in 35-37 ℃;
Hepatitis A virus gene group positive chain RNA in B, the extraction cell;
C, design of primers
Forward primer N1:
5’- TTGGGATTAGCGAGTATG-GGATGTTTCAGGAGTACAAGC-3’
Reverse primer N2:
5’- GACCTGGATAGGCTGTGTGAT-CTCTGATTGTTCTGTGACAGAC-3’
Forward primer M1:5 '-TTGGGATTAGCGAGTATG-3 '
Reverse primer M2:5 '-GACCTGGATAGGCTGTGTGAT-3 '
Forward primer N3:5 '-ATTACAGGAGCAACTGATGTAG-3 ';
D, detection hepatitis A virus (HAV) strand RNA intermediate
(1), reverse transcription reaction
In centrifuge tube, add following reagent successively:
10 * reverse transcription damping fluid, 2.5～5.0 μ l
DNTP mixture (2.5～10mmol/L) 1.25～10 μ l
Forward primer N1 (5～60 μ mol/L) 1.0～5 μ l
RNase inhibitor (40u/ μ l) 1.0～2.0 μ l
Reversed transcriptive enzyme (4u/ μ l) 1.0～2.0 μ l
Hepatitis A virus (HAV) RNA 15.0～30.0 μ l
Nuclease free water is supplemented to total reaction volume 25～50 μ l
Reacted 90～120 minutes down at 37～42 ℃; Obtaining reverse transcription reaction product was boiled 45～75 minutes;
(2), first round polymerase chain reaction
In centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 10.0～15.0 μ l
DNTP mixture (2.5～10mmol/L) 2.0～8.0 μ l
Reverse primer N2 (5～60 μ mol/L) 1.0～5.0 μ l
Forward primer M1 (5～60 μ mol/L) 1.0～5.0 μ l
Archaeal dna polymerase (5u/ μ l) 0.5～1.0 μ l
Reverse transcription reaction product 10.0～15.0 μ l
Nuclease free water is supplemented to total reaction volume 100～150 μ l
94 ℃ of preheatings 20～30 minutes, afterwards 94 ℃ of sex change 1～2 minute, 52～55 ℃ of annealing 1～2 minute, extended 1.5～2 minutes at 72 ℃, after the circulation like this 30～35 times, extended 20 minutes wheel polymerase chain (PCR) reaction product of winning again at 72 ℃;
(3), second take turns the polymerase chain reaction
In centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 5.0～7.5 μ l
DNTP mixture (2.5～10mmol/L) 1.0～4.0 μ l
Primer N3 (5～60 μ mol/L) 0.5～2.5 μ l
Primer M2 (5～60 μ mol/L) 0.5～2.5 μ l
Archaeal dna polymerase (5u/ μ l) 0.25～0.5 μ l
First round PCR product (1～10 times of dilution) 1.0～2.0 μ l
Nuclease free water is supplemented to total reaction volume 50～75 μ l
94 ℃ of preheatings 20～30 minutes, afterwards 94 ℃ of sex change 1～2 minute,, extended 1.5～2 minutes at 72 ℃ 52～55 ℃ of annealing 1～2 minute, so circulate after 30～35 times; Extended 20 minutes at 72 ℃ again, get second and take turns polymerase chain (PCR) reaction product;
E, electrophoretic analysis and titre are calculated
Take turns polymerase chain reaction (PCR) product with 1.5% concentration sepharose to second and carry out electrophoretic analysis, the positive result of DNA specific band person of 351bp length occurs, calculate titre with the Reed-Muench formula.
Described forward primer N1:
5 '- TTGGGATTAGCGAGTATGAmong-the GGATGTTTCAGGAGTACAAGC-3 ', non-line part is 2342 Nucleotide of the 2322nd Nucleotide to the of hepatitis A virus (HAV) HM-175 pnca gene group sequence, and the line part is a label construction, and neither complementation is also inequality with hepatitis A virus gene group sequence.
Described reverse primer N2:
5 '- GACCTGGATAGGCTGTGTGATNon-line part and 2970 Nucleotide complementations of the 2948th Nucleotide to the of hepatitis A virus (HAV) HM-175 pnca gene group sequence among-the CTCTGATTGTTCTGTGACAGAC-3 ', the line part is another label construction.
Described forward primer M1:5 '-TTGGGATTAGCGAGTATG-3 ' is consistent with label construction sequence among the primer N1.
Described reverse primer M2:5 '-GACCTGGATAGGCTGTGTGAT-3 ' is consistent with label construction sequence among the primer N2.
Described forward primer N3:5 '-ATTACAGGAGCAACTGATGTAG-3 ' is 2662 Nucleotide of the 2641st Nucleotide to the of hepatitis A virus (HAV) HM-175 pnca gene group sequence.
When reverse transcription, use primer N1; In first round polymerase chain reaction, use primer N2 and M1, expection product length is 677bp; Take turns in the polymerase chain reaction second, use primer N2 and M3, expection product length is 351bp.
The present invention compared with prior art has following advantage and effect:
1, in the present invention, the existence of infectious hepatitis A virus (HAV) is by chain specificity reverse transcriptase polymerase chain reaction method, and the mark strand RNA intermediate that the detection hepatitis A virus (HAV) occurs when duplicating is determined.It has broken through the limitation that existing reverse transcriptase polymerase chain reaction can not be distinguished infectious hepatitis A virus (HAV) and inactivation virus, the step that has overcome the cell culture method existence simultaneously is numerous and diverse, consuming time longer, often cause the vaccine stock phase long, shorten deficiencies such as vaccine validity period, directly detect the mark of hepatitis A virus (HAV), for the existence of infectious hepatitis A virus (HAV) provides reliable direct evidence.
2, when measuring the infection titer of live hepatitis a vaccines, compare with conventional cell culture method " Chinese biological goods rules " (2000 editions), cell cultures/chain specificity reverse transcriptase polymerase chain reaction method has following advantage: a, virus incubation time in cell can be foreshortened to about eight days by about one month, has shortened sense cycle greatly.B, in the virus culture process, need not replace and keep liquid, reduce the probability of polluting, reduce cell post-processing step such as multigelation, ultrasonic in the ordinary method, reduce workload, effectively increase work efficiency, do not need to detect antigen, avoid anti-hepatitis A antibody and two anti-uses with immunological method.C, can provide viral genetic information by order-checking to the polymerase chain reaction product.
Table 1 provides the virus infection titer that detects Attenuated HAV sample Ref with the present invention
Table 1.
Extent of dilution Detected result of the present invention
A B C D
10 -5 10 -6 10 -7 10 -8 + + - - + + + - + + + - + + + -
Annotate: "+" is positive, and "-" is negative.
Table 2 provides the virus infection titer that detects vaccine sample Ref with conventional cell culture method
Specifically undertaken by " Chinese biological goods rules " (2000 editions).The KMB17 cell that with generation was the 20th generation divides kind in 18 little square vase (25cm with 1: 3 ratio 2) in, grow up to fresh individual layer two days later.Attenuated Hepatitis A Vaccine,Live sample Ref is made 10 times of serial dilutions, get 10 -5, 10 -6, 10 -7With 10 -8Extent of dilution is seeded on the KMB17 cell, each extent of dilution inoculation 4 bottles of cell (A, B, C and D), and every bottle of cell inoculation virus liquid 1.0ml adsorbs to add after 2 hours and keeps liquid, cultivates in 35, changes liquid weekly once.Other two bottles of cells add 1mlPBS, are made as negative control.Harvested cell after 28 days, through multigelation, virus antigen is extracted in ultrasonic back, detects the hepatitis A virus (HAV) antigen protein with the ELISA method, the results are shown in Table 2.Calculating titre with the Reed-Muench formula is 10 7.50CCID 50/ ml.
Table 2.
Extent of dilution Conventional cell culture method detected result
A B C D
10 -5 10 -6 10 -7 10 -8 + + + - + + + - + + + - + + + -
Annotate: "+" is positive, and "-" is negative.
By the table in as can be known: detect hepatitis A Ref sample with the present invention, infection titer is 7.33CCID 50/ ml.Detect hepatitis A Ref sample with conventional cell culture method, infection titer is 7.50CCID 50/ ml.The result of two kinds of method detections is very approaching.
Description of drawings
Fig. 1 is for using the detected hepatitis A virus (HAV) strand RNA intermediate of invention;
Fig. 2 is for using the detected hepatitis A virus (HAV) strand RNA intermediate of invention.
Among Fig. 1, M:DNA Marker DL2000; C; Negative control; Road 1 to road 4:10 -5Dilution sample A, B, C and D; Road 5 to road 8:10 -6Dilution sample A, B, C and D.
Among Fig. 2, M:DNA Marker DL2000; C: negative control; Road 1 to road 4:10 -8Dilution sample A, B, C and D; Road 5 to road 8:10 -7Dilution sample A, B, C and D.
As seen from the figure: 10 -5, 10 -6Dilution sample A, B, C and D are positive entirely, and 10 -7Dilution sample A is negative, B, and C and D are positive, and 10 -8Dilution sample A, B, C and D are negative entirely.
Embodiment
Embodiment 1
1, virus culture
KMB17 cell branch is planted in little square vase, and cultivation grows up to fresh individual layer two days later; Attenuated HAV sample Ref is made 10 times of serial dilutions, get 10 -5, 10 -6, 10 -7With 10 -8Extent of dilution is seeded on the KMB17 cell, 4 bottles of cells of each extent of dilution inoculation, and every bottle of cell inoculation virus liquid 1.0ml adsorbs to add after 2 hours and keeps liquid, in 35 ℃ of cultivations.Other two bottles of cells add the 1ml phosphate buffered saline buffer, are made as negative control.
2, the extraction of hepatitis A virus (HAV) RNA in the cell
Cultivate after 8 days, blot keeping liquid in the culturing bottle, every bottle adds 2.5mlTRIzol reagent and carries out cracking, even lysate branch is installed in 2 1.5ml pipes, one pipe is used for subsequent experimental at once, one guarantees that to be stored in-70 ℃ standby, operates by the reagent operational manual subsequently, and every bottle of cell can obtain 50 μ lRNA samples.
3, extract hepatitis A virus gene group positive chain RNA in the cell;
4, design of primers
Forward primer N1:
5’- TTGGGATTAGCGAGTATG-GGATGTTTCAGGAGTACAAGC-3’
Reverse primer N2:
5’- GACCTGGATAGGCTGTGTGAT-CTCTGATTGTTCTGTGACAGAC-3’
Forward primer M1:5 '-TTGGGATTAGCGAGTATG-3 '
Reverse primer M2:5 '-GACCTGGATAGGCTGTGTGAT-3 '
Forward primer N3:5 '-ATTACAGGAGCAACTGATGTAG-3 ';
5, detect hepatitis A virus (HAV) strand RNA intermediate
(1), reverse transcription reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * reverse transcription damping fluid, 2.5 μ l
DNTP mixture (5mmol/L) 2 μ l
Forward primer N1 (5～60 μ mol/L) 1 μ l
RNase inhibitor (40u/ μ l) 1 μ l
Reversed transcriptive enzyme (4u/ μ l) 2 μ l
Hepatitis A virus (HAV) RNA 15 μ l
Nuclease free water is supplemented to total reaction volume 25 μ l
Reacted 90 minutes down at 37 ℃; Obtaining reverse transcription reaction product was boiled 45 minutes;
(2), first round polymerase chain reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 10 μ l
DNTP mixture (10mmol/L) 2 μ l
Reverse primer N2 (5～60 μ mol/L) 1 μ l
Forward primer M1 (5～60 μ mol/L) 1 μ l
Archaeal dna polymerase (5u/ μ l) 0.5 μ l
Reverse transcription reaction product 15 μ l
Nuclease free water is supplemented to total reaction volume 100 μ l
94 ℃ of preheatings 20 minutes, afterwards 94 ℃ of sex change 1 minute,, extended 1.5 minutes at 72 ℃ 52 ℃ of annealing 1 minute, so circulate after 30 times, extended 20 minutes wheel polymerase chain (PCR) reaction product of winning again at 72 ℃;
(3), second take turns the polymerase chain reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 5 μ l
DNTP mixture (10mmol/L) 1 μ l
Primer N3 (5～60 μ mol/L) 0.5 μ l
Primer M2 (5～60 μ mol/L) 0.5 μ l
Archaeal dna polymerase (5u/ μ l) 0.5 μ l
First round PCR product (1～10 times of dilution) 2.0 μ l
Nuclease free water is supplemented to total reaction volume 50 μ l
94 ℃ of preheatings 20 minutes, afterwards 94 ℃ of sex change 1 minute,, extended 1.5 minutes at 72 ℃ 52 ℃ of annealing 1 minute, so circulate after 30 times; Extended 20 minutes at 72 ℃ again, get second and take turns polymerase chain (PCR) reaction product;
E, electrophoretic analysis and titre are calculated
Get 8 μ l second and take turns the polymerase chain reaction product and carry out electrophoretic analysis (with 1.5% concentration sepharose), deposition condition is 100V, 30 minutes, and to observe down and take a picture in ultraviolet lamp, electrophoresis result is seen Fig. 1 and Fig. 2.The positive result of 351bp length DNA specific band person occurs, statistics sees Table 1.Calculating titre with the Reed-Muench formula is 10 7.33CCID 50/ ml.This result extremely approaches with conventional cell culture method measured value 10 7.50CCID 50/ ml.
Embodiment 2
1,2,3 and 4 steps are with embodiment 1;
5, detect hepatitis A virus (HAV) strand RNA intermediate
(1), reverse transcription reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * reverse transcription damping fluid, 5 μ l
DNTP mixture (3mmol/L) 4 μ l
Forward primer N1 (5～60 μ mol/L) 2 μ l
RNase inhibitor (40u/l) 2 μ l
Reversed transcriptive enzyme (4u/ μ l) 2 μ l
Hepatitis A virus (HAV) RNA 30 μ l
Nuclease free water is supplemented to total reaction volume 50 μ l
Reacted 120 minutes down at 42 ℃; Obtaining reverse transcription reaction product was boiled 75 minutes;
(2), first round polymerase chain reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 15 μ l
DNTP mixture (3mmol/L) 4 μ l
Reverse primer N2 (5～60 μ mol/L) 2 μ l
Forward primer M1 (5～60 μ mol/L) 2 μ l
Archaeal dna polymerase (5u/ μ l) 1 μ l
Reverse transcription reaction product 12 μ l
Nuclease free water is supplemented to total reaction volume 150 μ l
94 ℃ of preheatings 30 minutes, afterwards 94 ℃ of sex change 2 minutes,, extended 2 minutes at 72 ℃ 55 ℃ of annealing 2 minutes, so circulate after 35 times, extended 20 minutes wheel polymerase chain (PCR) reaction product of winning again at 72 ℃;
(3), second take turns the polymerase chain reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 7 μ l
DNTP mixture (3mmol/L) 2 μ l
Primer N3 (5～60 μ mol/L) 1 μ l
Primer M2 (5～60 μ mol/L) 1 μ l
Archaeal dna polymerase (5u/ μ l) 0.5 μ l
First round PCR product (1～10 times of dilution) 2 μ l
Nuclease free water is supplemented to total reaction volume 70 μ l
94 ℃ of preheatings 30 minutes, afterwards 94 ℃ of sex change 2 minutes,, extended 1.5 minutes at 72 ℃ 55 ℃ of annealing 2 minutes, so circulate after 35 times, extended 20 minutes at 72 ℃ again, second take turns polymerase chain (PCR) reaction product;
6, take turns polymerase chain reaction (PCR) product with 1.5% concentration sepharose to second and carry out electrophoretic analysis, the positive result of DNA specific band person of 351bp length occurs, calculating titre with the Reed-Muench formula is 7.33CCID 50/ ml.This result extremely approaches with conventional cell culture method measured value 10 7.50CCID 50/ ml.
Embodiment 3
1,2,3 and 4 steps are with embodiment 1;
5, detect hepatitis A virus (HAV) strand RNA intermediate
(1), reverse transcription reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * reverse transcription damping fluid, 3.5 μ l
DNTP mixture (8mmol/L) 2 μ l
Forward primer N1 (5～60 μ mol/L) 1 μ l
RNase inhibitor (40u/ μ l) 1 μ l
Reversed transcriptive enzyme (4u/ μ l) 2 μ l
Hepatitis A virus (HAV) RNA 15 μ l
Nuclease free water is supplemented to total reaction volume 35 μ l
Reacted 100 minutes down at 40 ℃; Obtaining reverse transcription reaction product was boiled 55 minutes;
(2), first round polymerase chain reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 12 μ l
DNTP mixture (6mmol/L) 4 μ l
Reverse primer N2 (5～60 μ mol/L) 3 μ l
Forward primer M1 (5～60 μ mol/L) 3 μ l
Archaeal dna polymerase (5u/ μ l) 0.8 μ l
Reverse transcription reaction product 12 μ l
Nuclease free water is supplemented to total reaction volume 120 μ l
94 ℃ of preheatings 15 minutes, afterwards 94 ℃ of sex change 1 minute,, extended 2 minutes at 72 ℃ 54 ℃ of annealing 1 minute, so circulate after 36 times, extended 20 minutes wheel polymerase chain (PCR) reaction product of winning again at 72 ℃;
(3), second take turns the polymerase chain reaction
In the 0.5ml of nuclease free centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 6 μ l
DNTP mixture (7mmol/L) 3 μ l
Primer N3 (5～60 μ mol/L) 1.5 μ l
Primer M2 (5～60 μ mol/L) 1.5l
Archaeal dna polymerase (5u/ μ l) 0.4 μ l
First round PCR product (1～10 times of dilution) 2.0 μ l
Nuclease free water is supplemented to total reaction volume 60 μ l
94 ℃ of preheatings 25 minutes, afterwards 94 ℃ of sex change 1 minute,, extended 1.5 minutes at 72 ℃ 54 ℃ of annealing 1 minute, so circulate after 37 times; Extended 20 minutes at 72 ℃ again, get second and take turns polymerase chain (PCR) reaction product;
6, take turns polymerase chain reaction (PCR) product with 1.5% concentration sepharose to second and carry out electrophoretic analysis, the positive result of DNA specific band person of 351bp length occurs, calculating titre with the Reed-Muench formula is 7.50CCID 50/ ml.This result with conventional cell culture method measured value 10 7.50CCID 50/ ml is identical.

Claims (2)

1, a kind of fast, the method for Sensitive Detection virus infection titer of attenuated live hepatitis A vaccine, it is characterized in that comprising the following steps:
A, virus culture
The KMB17 cell is implanted in the little square vase, be cultured to grow up to fresh individual layer after, the Attenuated HAV sample is made 10 times of serial dilutions, getting extent of dilution is 10 -4-10 -9Viral liquid be seeded on this cell monolayer, adsorb after 1～2 hour, add and keep liquid, cultivated 5-8 days in 35-37 ℃;
Hepatitis A virus gene group positive chain RNA in B, the extraction cell;
C, design of primers
Forward primer N1:
5’- TTGGGATTAGCGAGTATG-GGATGTTTCAGGAGTACAAGC-3’
Reverse primer N2:
5’- GACCTGGATAGGCTGTGTGAT-CTCTGATTGTTCTGTGACAGAC-3’
Forward primer M1:5 '-TTGGGATTAGCGAGTATG-3 '
Reverse primer M2:5 '-GACCTGGATAGGCTGTGTGAT-3 '
Forward primer N3:5 '-ATTACAGGAGCAACTGATGTAG-3 ';
D, detection hepatitis A virus (HAV) strand RNA intermediate
(1), reverse transcription reaction
In centrifuge tube, add following reagent successively:
10 * reverse transcription damping fluid, 2.5～5.0 μ l
DNTP mixture (2.5～10mmol/L) 1.25～10 μ l
Forward primer N1 (5～60 μ mol/L) 1.0～5 μ l
RNase inhibitor (40u/ μ l) 1.0～2.0 μ l
Reversed transcriptive enzyme (4u/ μ l) 1.0～2.0 μ l
Hepatitis A virus (HAV) RNA 15.0～30.0 μ l
Nuclease free water is supplemented to total reaction volume 25～50 μ l
Reacted 90～120 minutes down at 37～42 ℃; Obtaining reverse transcription reaction product was boiled 45～75 minutes;
(2), first round polymerase chain reaction
In centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 10.0～15.0 μ l
DNTP mixture (2.5～10mmol/L) 2.0～8.0 μ l
Reverse primer N2 (5～60 μ mol/L) 1.0～5.0 μ l
Forward primer M1 (5～60 μ mol/L) 1.0～5.0 μ l
Archaeal dna polymerase (5u/ μ l) 0.5～1.0 μ l
Reverse transcription reaction product 10.0～15.0 μ l
Nuclease free water is supplemented to total reaction volume 100～150 μ l
94 ℃ of preheatings 20～30 minutes, afterwards 94 ℃ of sex change 1～2 minute, 52～55 ℃ of annealing 1～2 minute, extended 1.5～2 minutes at 72 ℃, after the circulation like this 30～35 times, extended 20 minutes wheel polymerase chain (PCR) reaction product of winning again at 72 ℃;
(3), second take turns the polymerase chain reaction
In centrifuge tube, add following reagent successively:
10 * PCR damping fluid, 5.0～7.5 μ l
DNTP mixture (2.5～10mmol/L) 1.0～4.0 μ l
Primer N3 (5～60 μ mol/L) 0.5～2.5 μ l
Primer M2 (5～60 μ mol/L) 0.5～2.5 μ l
Archaeal dna polymerase (5u/ μ l) 0.25～0.5 μ l
First round PCR product (1～10 times of dilution) 1.0～2.0 μ l
Nuclease free water is supplemented to total reaction volume 50～75 μ l
94 ℃ of preheatings 20～30 minutes, afterwards 94 ℃ of sex change 1～2 minute,, extended 1.5～2 minutes at 72 ℃ 52～55 ℃ of annealing 1～2 minute, so circulate after 30～35 times; Extended 20 minutes at 72 ℃ again, get second and take turns polymerase chain (PCR) reaction product;
E, electrophoretic analysis and titre are calculated
Take turns polymerase chain reaction (PCR) product with 1.5% concentration sepharose to second and carry out electrophoretic analysis, the positive result of DNA specific band person of 351bp length occurs, calculate titre with the Reed-Muench formula.
2, method according to claim 1 is characterized in that described:
Forward primer N1:
5 '- TTGGGATTAGCGAGTATGAmong-the GGATGTTTCAGGAGTACAAGC-3 ', non-line part is 2342 Nucleotide of the 2322nd Nucleotide to the of hepatitis A virus (HAV) HM-175 pnca gene group sequence, and the line part is a label construction, and neither complementation is also inequality with hepatitis A virus gene group sequence;
Reverse primer N2:
5 '- GACCTGGATAGGCTGTGTGATNon-line part and 2970 Nucleotide complementations of the 2948th Nucleotide to the of hepatitis A virus (HAV) HM-175 pnca gene group sequence among-the CTCTGATTGTTCTGTGACAGAC-3 ', the line part is another label construction;
Forward primer M1:5 '-TTGGGATTAGCGAGTATG-3 ' is consistent with label construction sequence among the primer N1;
Reverse primer M2:5 '-GACCTGGATAGGCTGTGTGAT-3 ' is consistent with label construction sequence among the primer N2;
Forward primer N3:5 '-ATTACAGGAGCAACTGATGTAG-3 ' is 2662 Nucleotide of the 2641st Nucleotide to the of hepatitis A virus (HAV) HM-175 pnca gene group sequence.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652590A (en) * 2018-12-18 2019-04-19 云南沃森生物技术股份有限公司 A kind of strand RNA molecular biology for detection of hepatitis A virus and application
CN111500772A (en) * 2020-04-28 2020-08-07 中国食品药品检定研究院 Rapid hepatitis A vaccine virus titer detection method based on PMA-qRT-PCR method
CN112226534A (en) * 2020-09-08 2021-01-15 广东省农业科学院动物卫生研究所 Primer group and kit for detecting novel coronavirus proliferation activity and application of primer group and kit

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Publication number Priority date Publication date Assignee Title
CN1108386C (en) * 1999-06-15 2003-05-14 浙江省医学科学院普康生物技术公司 Reverse transcription of attenuated live hepatitis A vaccine titre as one polymerase chain reaction detecting process
EP1552022B1 (en) * 2002-06-12 2013-01-23 Novartis Vaccines and Diagnostics, Inc. Identification of oligonucleotides for the capture, detection and quantitation of hepatitis a viral nucleic acid

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652590A (en) * 2018-12-18 2019-04-19 云南沃森生物技术股份有限公司 A kind of strand RNA molecular biology for detection of hepatitis A virus and application
CN109652590B (en) * 2018-12-18 2022-10-21 云南沃森生物技术股份有限公司 Hepatitis A virus negative strand RNA molecular biological detection method and application
CN111500772A (en) * 2020-04-28 2020-08-07 中国食品药品检定研究院 Rapid hepatitis A vaccine virus titer detection method based on PMA-qRT-PCR method
CN111500772B (en) * 2020-04-28 2022-10-18 中国食品药品检定研究院 Rapid hepatitis A vaccine virus titer detection method based on PMA-qRT-PCR method
CN112226534A (en) * 2020-09-08 2021-01-15 广东省农业科学院动物卫生研究所 Primer group and kit for detecting novel coronavirus proliferation activity and application of primer group and kit

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